Abstract: :
Purpose: Increased IOP is a known risk factor for primary open–angleglaucoma (POAG).The aqueous humor of patients with normal pressureglaucoma and POAG contains increased concentration of sCD44,the ectodomain fragment of CD44. CD44 is a receptor of hyaluronicacid(HA) whereas sCD44 is a neurotoxin to retinal ganglion cellsin vitro. In the present study, we determined the binding affinityof standard sCD44 and hypo– phosphorylated sCD44 to HA.Methods: sCD44 was isolated from human sera by urea solubilizationand immunoprecipitation and subjected to in vitro hypo–phosphorylationby incubation with 30 units of alkaline phosphatase for 1 hourat 37C and repeated. Highly purified HA was immobilized on EAH–Sepharose,packed in mini–columns, and graded amounts of standardand hypo–phosphorylated sCD44 were loaded on the columnand eluted at operating pressures of 0 to 40 mmHg. The columnwas rinsed and bound sCD44 was eluted with glycine. Fractionswere concentrated and analyzed by ELISA. Scatchard plots wereconstructed using Graphpad Prism 4.0.Results: The dissociationconstant of hypo–phosphorylated sCD44 was 0.0868 nM toHA which was five–fold greater than the dissociation constantof standard sCD44. As the operating pressure of the column wasincreased to 40 mmHg, the dissociation constant increased sothat less sCD44 bound to the HA. Additionally, though the maximumbinding was comparable (Vmax), standard sCD44 bound 44 umol/molHA and the hypo–phosphorylated sCD44 bound 60 umol/molHA. Conclusions: An intriguing hypothesis could be drawn fromthese results. HA in the vitreous acts as a reservoir, an extracellular"dump" site, for sCD44. As IOP increases, the vitreous releasesthe neurotoxic sCD44, particularly hypo–phosphorylatedsCD44, which could explain the risk factor of increased IOPin POAG.